out in a 10-μL total reaction volume with 500 nM of each primer and 100 nM of fluorescent probe using PerfeCta qPCR FastMix (Quanta BioSciences, Gaithersburg, Maryland). Thermocycling consisted of 2 minutes at 45°C for uracil-N-glycosylase incubation, 30 seconds at 95°C for hot-start activation, and 40 amplification cycles of 2 seconds at 95°C and 20 seconds at 60°C followed by fluorescence acquisition in a LightCycler 480 instrument (Roche Applied Science). Crossing-point determination by second derivative maximum was carried out with LightCycler 480 software, release 1.3. Relative abundances of SLC1A1 messenger RNA were then calculated using the efficiency-corrected comparative threshold method and log2-transformed.27 Efficiency calculations were performed with 2-fold serially diluted pooled complementary DNA (range, 30-0.47 ng per reaction). Amplicon size and assay specificity were verified by agarose electrophoresis. No-template controls did not display detectable amplification (data not shown). All experiments were done after the specimen code was broken and they were thus unblinded. The entire data set has been uploaded to the Stanley Medical Research Institute data bank (http://www.stanleyresearch.org/brain).
